Comprehensive Western Blot Analysis

Western blotting was performed as described previously [30 (link), 35 (link)], and MT expression was detected using a modified western blotting protocol [24 (link)]. Primary antibodies against the following were used: 3-nitrotyrosine (3-NT; 1 : 1,000; Millipore, Billerica, MA, USA); Nrf2 (1 : 1,000; Abcam, Cambridge, MA, USA); α-smooth muscle actin (α-SMA; 1 : 1,000; Abcam); connective tissue growth factor (CTGF; 1 : 1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); fibronectin (1 : 1,000; Abcam); tumor necrosis factor α (TNF-α; 1 : 1,000; Abcam); NACHT, LRR, and PYD domain-containing protein 3 (NLRP3; 1 : 1,000; Abcam); NAD(P)H:quinone oxidoreductase 1 (NQO1; 1 : 1,000; Santa Cruz Biotechnology); superoxide dismutase-2 (SOD-2; 1 : 5,000; Santa Cruz Biotechnology); catalase (CAT; 1 : 5,000; Santa Cruz Biotechnology); β-actin (1 : 8,000; Santa Cruz Biotechnology); and MT (1 : 1,000; DakoCytomation, Carpinteria, CA, USA).
The protein content was determined by measuring the gray values of bands using Image Lab (Bio-Rad).